Method for extracting cocoa procyanidins

ABSTRACT

A cocoa extract which is rich in procyanidin monomers and oligomers is made by extracting de-fatted, unroasted, unfermented cocoa beans with organic solvents. The yield of procyanidins in an extract varies with type of solvent used, reaction temperature, reaction pH and whether or not the solvent is an aqueous solution. Extraction parameters can be optimized to increase procyanidin yield, and different conditions result in the preferential extraction of the higher or lower oligomers. A preferred extraction method is counter-current extraction method.

FIELD OF THE INVENTION

[0001] This invention is directed to improved methods for the extractionof cocoa procyanidin monomers and oligomers from the cocoa solids.

[0002] It is known that regular consumption of dietary polyphenols,commonly found in a variety of fruits and vegetables, is beneficial. Redwine, green tea and cocoa have all been identified as being rich inpolyphenols, and the regular consumption red wine and green tea haveboth been shown to be inversely associated with heart disease deaths inindustrialized countries.

BACKGROUND OF THE INVENTION

[0003] It is well-known that the polyphenols of cocoa contributesignificantly to the development of flavour in the fermented and roastedcocoa bean. Astringent and bitter flavors in cocoa have beentraditionally associated with the presence of xanthine alkaloids andpolyphenols in the cocoa beans. For this reason, various methods havebeen developed over the years to extract the cocoa polyphenols to verifytheir presence, to quantify their amounts, and to identify them. Thecocoa polyphenols are primarily cocoa procyanidins. However, noextraction method has thus far been optimized to yield extracts high incocoa procyanidins.

[0004] It is now known that the cocoa procyanidin oligomers show a clearrelationship between structure and function, meaning that individualoligomers, or fractions containing several oligomers of a similar size,show specific biological functions which are not affected by otheroligomers. Thus, it is important to ensure that the extraction procedureutilized not only results in the highest possible solubilization of thecocoa polyphenols, but that it is effective at extracting all the cocoapolyphenol oligomers present in the bean.

[0005] The extraction of cocoa beans using water or an organic solvent,or a mixture of water and an organic solvent, has been used to removethe xanthine alkaloids (predominantly caffeine and theobromine) andother soluble constituents of the cocoa bean which impart a bitter,disagreeable flavor. Included among these bitter-tasting solubleconstituents are the procyanidins.

[0006] U.S. Pat. No. 1,750,795 (issued to Defren in 1926), discloses aprocess for removing the “greater part of the soluble bitterconstituents of the beans” by soaking the beans in water at 60° C. andthen discarding the water and roasting the beans.

[0007] Hot water treatments have been used to remove the xanthines inorder to provide stimulant-free cocoa beans. See U.S. Pat. No. 4,407,834“Detheobromination of Cocoa” (issued to Chiovini et al., Jun. 28, 1983)and U.S. Pat. No. 4,755,391 “Removal of Methylxanthines from CacaoMaterials” (issued to Chiovini et al, Jul. 5, 1988).

[0008] Cocoa extracts have been prepared by extracting cocoa solids,prepared from fermented, conventionally roasted cocoa beans or cocoanibs, with water and/or alcohols. Osakabe et al. used ethanol, eitherabsolute or aqueous at greater than 40% v/v with deionized water atambient temperature to prepare a crude polyphenol extract. See JP946-64717 “Food or Beverage Product for Preventing Gastric Ulcers” (Oct.4, 1995).

[0009] Zieglader et al. used methanol to extract the polyphenols fromcocoa beans at ambient temperature. See “Antioxidative Effects of Cocoa”(Rev. Choc.Confect. Bak, 8:3-6, 1983) which discloses the preparation ofa methanol extract. The extract contained “monomer tannin precursors(catechins, anthocyanidins and their soluble condensates)” and that isused as an additive for oil to preserve it from oxidation. Griffiths etal. used methanol extracts, again obtained at ambient temperatures, toobtain polyphenol extracts from ripe cocoa nibs, which were used tostudy the characterization of plant polyphenols in cocoa and otherplants. See “A Comparative Study of the Seed Polyphenols of the GenusTheobroma ”, (Biochemical J. 74: 362-365, 1960). Rigaud et al. made anextract from lyophilized cocoa beans and grape seeds, and noted that theuse of methanol as a solvent precluded the presence of the higheroligomers. See “Normal-Phase High-Performance Liquid ChromatographicSeparation of Procyanidins from Cacao Beans and Grape Seeds”, (J.Chromatography 654:255-60, 1993). Jalal & Collin prepared extracts fromdifferent parts of the cocoa plant in order to analyze the polyphenolspresent in each part of the plant. The extraction was carried out using70% cold methanol, followed by ethyl acetate. See “Polyphenols of MaturePlant, Seedling and Tissue Cultures of Theobroma Cacao” (Phytochemistry,16:1377-1380, 1977).

[0010] Acetone/water has also be used for cocoa bean extractions.Clapperton et al. report the extraction of defatted cocoa powder madefrom fermented cocoa beans using cold 70% acetone. See “Polyphenols andCocoa Flavor, Groupe Polyphenols,” (XVI^(th) Intern. Conf., Lisbon,Portugal, July 13-16, 1992). Rigaud combines a first extraction withethanol with a second extraction using a 60% acetone/water mixture. See“Normal-Phase High-Performance Liquid Chromatographic Separation ofProcyanidins from Cacao Beans and Grape Seeds” (J. Chromatography,654:255-60, 1993).

[0011] Traditionally, cocoa beans are treated and processed in such away as to minimize the bitter taste which the polyphenols impart to thebeans, and this results in a reduction of the polyphenol content of thebean. The two ways in which the polyphenol content is significantlyreduced are fermentation of cocoa beans in their husks and roasting thefermented cocoa beans in order to crack their husks and aid thede-hulling process. Zieglader at al. report a loss of antioxidantactivity in extracts from cocoa beans which are fermented compared toextracts from unfermented beans, and correlate this reduction inantioxidant potential with a reduced amount of polyphenols in theextracts of fermented beans. All the previously discussed extractionmethods were carried out using fermented beans, and in many cases thebeans were also roasted. Therefore, the procyanidin yields are muchlower than those found in extracts from unfermented beans.

[0012] It has been a common practice to follow an initial solventextraction step with an ethyl acetate extraction step. See Forsyth &Roberts, “Cacao Polyphenolic Substances: 3. The Structure of CacaoLeucocyanidin 1” Biochem. J. 74, 374-378, 1960; Thompson et al., “PlantProcyanidins. Part I. Introduction; the Isolation, Structure andDistribution in Nature of Plant Procyanidins” J. Chem. Soc. Perkin I,Vol. 11, 1387-99, 1972; Jalal & Collins, “Polyphenols of Mature Plant,Seedling and Tissue Cultures of Theobroma Cacao”, Phytochemistry,16:1377-1380, 1977, Porter et al. “Flavans and Proanthocyanidins”Chapter Two in “The Flavonoids”, Ed., J. B. Harborne, Chapman and HallLtd., London, 1988; U.S. Pat. No. 5,554,645 (issued Oct. 3, 1994 toRomanczyk et al). This has the effect of producing an extract whichcontains the monomers and lower oligomers and none, or very little, ofthe higher oligomers. See Lea A. “The Phenolics of Ciders: Oligomericand Polymeric Procyanidins”, J. Sci. Fd Agric. 29, 471-477, 1978.

[0013] Whether or not the cocoa beans are defatted prior to extractionhas an impact upon the yield of polyphenols extracted. If the beans arenot defatted, the fat in the beans interferes with the solubilizingactivity of the solvent, and the polyphenol yields are considerablyreduced (Lazarus et al., “Flavonoids and Other Polyphenols” in Methodsin Enzymology series. Edited by Lester Packer, Academic Press, New York,in press).

[0014] Cocoa extracts have been prepared from cocoa solids prepared fromunfermented or fermented sun dried cocoa beans. The beans were ground,defatted and extracted. A 70% acetone/30% deionized water mixture wasused, followed by a 70% methanol extraction and two chloroformextractions. Then follows an extraction with ethyl acetate, the additionof water, and the removal of the ethyl acetate. Alternatively, the beanswere extracted with 70% acetone. In both cases the extractions werecarried out at room temperature and the aqueous extracts werefreeze-dried. See U.S. Pat. No. 5,554,645 (issued Oct. 3, 1994 to L.Romanczyk et al.).

[0015] Furthermore, cocoa extracts can be prepared from partiallydefatted cocoa solids that are prepared from cocoa beans which have notbeen roasted. The resulting cocoa solids, whether prepared fromfermented, underfermented, or unfermented cocoa nibs, have a highercocoa polyphenol content, i.e., cocoa procyanidin content, than cocoasolids prepared from conventionally roasted cocoa beans or cocoa nibs.See U.S. Pat. No. 6,015,913 (issued Jan. 18, 2000 to K. S. Kealey etal.).

[0016] Defatted, freeze-dried, unfermented cocoa beans contain about 2%xanthine alkaloids, and traditionally processed beans contain largeramounts. Theobromine is a degradation product of caffeine, and both arewell-known stimulants of the nervous system. Excessive xanthine alkaloidintake is not thought to be beneficial. Hence, in some cases, it may bedesirable to cocoa extracts free of the xanthine alkaloids.

[0017] Thus, there is a need for an improved process to extract cocoaprocyanidins from cocoa solids, and, in some cases, to decaffeinate anddetheobrominate the cocoa extracts.

SUMMARY OF THE INVENTION

[0018] The present invention provides an improved method for extractingcocoa polyphenols including cocoa procyanidins from partially or fullydefatted cocoa solids prepared from non-roasted cocoa beans. Theimproved process comprises a single extraction, at atmospheric pressureor under pressure, with a solvent selected from the group consisting ofan acidified organic solvent capable of solubilizing the polyphenols, oraqueous mixtures thereof. An edible acid is added to solvent oraqueous-solvent mixture in an amount sufficient to lower the pH fromabout 6.5 to about 2 to about 4. The cocoa solids are prepared fromunfermented or underfermented cocoa beans. The organic solvent may beselected from the group consisting of lower alkyl alcohols, lower alkylketones, and lower alkyl acetate. Suitable solvents include methanol,ethanol, isopropanol, acetone, methyl acetate, or ethyl acetate. Theaqueous solvent mixture preferably contains up to about 50% water byvolume. Preferred aqueous-solvent mixtures include isopropanol and about50-70% water, isopropanol and about 20% water which preferentiallyextracts monomers and oligomers up to and including hexamers, acetoneand about 80% to about 100% water, acetone and about 50% to about 70%water, or 100% ethanol which preferentially extracts cocoa procyanidinoligomers higher than the heptamers is enhanced.

[0019] The present invention also provides a method of extracting cocoapolyphenols including cocoa procyanidin monomers and oligomers frompartially defatted or fully defatted cocoa solids prepared from cocoabeans that have not been roasted. The method comprises the step ofextracting, at atmospheric pressure or under pressure, the cocoa solidswith a non-acidified organic solvent capable of solubilizing cocoapolyphenols or non-acidified aqueous mixtures thereof. Preferably, thecocoa beans are unfermented or underfermented cocoa beans. The beanstypically have a fermentation factor of 275 or less. The preferred beansinclude slaty, purple, or purple brown cocoa beans, or mixtures of slatyand purple cocoa beans, purple and brown cocoa beans, or slaty, purpleand brown cocoa beans. The unfermented or underfermented cocoa beanscontain at least about 1% up to about 15% by weight of total cocoaprocyanidins per gram of defatted cocoa solids, typically about 4-7%.The organic solvent may be selected from the group consisting of a loweralkyl alcohol, a lower alkyl ketone, and a lower alkyl acetate. Suitableorganic solvents are methanol, ethanol, isopropanol, acetone, methylacetate, or ethyl acetate. The aqueous mixture can contain up to about50% water by volume. Preferred solvents include 50-60% acetone, and 100%ethanol. The extraction can be carried out at a temperature from 0° C.up to the boiling point of the solvent or aqueous solvent mixture usedat the extraction pressure used. When the solvent is methanol, theextraction is preferably carried out at room temperature. When thesolvent is ethanol, the extraction is preferably carried out at about20° C. to about 50° C. When the solvent is isopropanol, the extractionis preferably carried out at about 70° C. When the solvent is anaqueous-acetone mixture, the extraction is preferably carried out atabout 50° C. up to the boiling point of the mixture.

[0020] In the above process, when an acidified solvent or solventmixture is used, a lower extraction temperature is preferred. When anon-acidified solvent or solvent mixture is used, a higher extractiontemperature can be used.

[0021] The present invention also provides a method for selectivelyextracting low molecular weight cocoa procyanidin oligomers frompartially or fully defatted cocoa solids, prepared from cocoa beans thathave not been roasted. The solvent used is a solvent whichpreferentially extracts the lower oligomers, e.g., methyl acetate orethyl acetate. Preferably, the cocoa beans are unfermented orunderfermented cocoa beans. The extraction is preferably carried out atabout 20° C. to about 50° C. The resulting cocoa extracts consistessentially of monomers, dimers and trimers,

[0022] The present invention also provides a method for selectivelyextracting higher molecular weight cocoa procyanidin oligomers frompartially defatted or fully defatted cocoa solids prepared from cocoabeans that have not been roasted. The method comprises the steps of (a)extracting the cocoa solids with ethyl acetate; (b) recovering theextracted cocoa solids; (c) extracting the recovered extracted cocoasolids with a solvent which is a good solvent for the higher oligomer,e.g., acetone and ethanol and mixtures there of with up to 50% water;(d) separating the cocoa solids from the cocoa extract; and (e)optionally drying the cocoa extract. The extraction is preferablycarried out at about 20° C. to about 50° C. Preferably the cocoa beansare unfermented or underfermented. The cocoa extracts consistsessentially of at least tetramers and higher oligomers.

[0023] The present invention further provides a continuous method forextracting, at atmospheric pressure or under pressure, cocoa polyphenolsincluding cocoa procyanidins from partially defatted or fully defattedcocoa solids using an organic solvent suitable for solubilizing cocoaprocyanidins, or mixtures thereof with water. The process comprises thesteps of (a) contacting a series of cells containing the cocoa solidswith a solvent flowing in a counter-current direction, (b) recoveringthe cocoa extract, and (c) drying the cocoa extract. Preferred solventsinclude lower alkyl alcohols, lower alkyl ketones, and lower alkylacetates. Suitable lower alkyl alcohols are methanol, ethanol, andisopropanol. A suitable lower alkyl ketones is acetone. Suitable loweralkyl acetates are methyl acetate and ethyl acetate. Preferably, thecocoa extract is recycled until substantially no more cocoa procyanidinsare extracted from the cocoa solids. When the solvent is the organicsolvent, the cocoa extract is dried by flashing off the solvent. Whenthe solvent is the aqueous-organic mixture, the cocoa extract is driedby flashing off the solvent before drying the aqueous extract. Theaqueous extracts can be dried by freeze drying, spray drying, flashdrying, or drum drying.

[0024] The present invention provides a method for recovering cocoaprocyanidins and theobromine from partially defatted or fully defattedcocoa solids, prepared from cocoa beans. The process comprises the stepsof (a) extracting the cocoa solids with ethanol at about 50° C. to about70° C., (b) chilling the ethanol extract at a temperature and for a timesufficient to precipitate out the theobromine, (c) separating theprecipitated out theobromine, and (d) removing the ethanol from extractto recover the dried cocoa procyanidins. Preferably, the beans areunfermented or underfermented cocoa beans that have not been roasted.The present invention also provides a method of minimizing the amount oftheobromine in the cocoa procyanidins extracted from partially defattedor fully defatted cocoa solids, prepared from unroasted, preferablyunfermented or underfermented, cocoa beans. The method comprises thesteps of (a) extracting the cocoa solids with isopropanol to produce analcoholic extract containing cocoa procyanidins and theobromine, (b)chilling the alcoholic extract at a temperature and for a timesufficient to precipitate out the theobromine, and (c) separating thetheobromine from the alcoholic extract. In the above processes, thechilling is carried out for up to about 12 hours at 0° C. to about 5° C.and the separating step is carried out by filtering the extract,centrifuging the extract, or by an adsorbing the theobromine on asuitable absorbent.

DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1(a) Graph showing the effect of temperature on yield of themonomer, pentamer and decamer using 100% ethanol as the extractionsolvent.

[0026]FIG. 1(b) Graph showing the effect of temperature on yield of themonomer, pentamer and decamer using 100% isopropanol as the extractionsolvent.

[0027]FIG. 1(c) Graph showing the effect of temperature on yield of themonomer, pentamer and decamer using 100% methanol as the extractionsolvent.

[0028]FIG. 2 Graph showing that acetone and ethanol are most effectivein extracting the higher oligomers.

[0029]FIG. 3 Graph showing the effects of fermentation and processing onprocyanidin yields using an acetone:water:acetic acid solvent as thestandard.

[0030]FIG. 4 Graph showing the effect of fermentation and processing onprocyanidin extraction with boiling water.

[0031]FIG. 5 A schematic diagram of the process of extractingpolyphenols from cocoa beans.

[0032]FIG. 6 A schematic diagram showing the process ofde-theobrominating a cocoa extract.

[0033]FIG. 7 A graph comparing the oligomeric profiles of an ethylacetate extraction of cocoa solids, and an acetone/water/acetic acidextract of the ethyl acetate-extracted solids

DETAILED DESCRIPTION OF THE INVENTION

[0034] Cocoa procyanidins can be obtained from several Theobroma cacaogenotypes by the procedures discussed hereinafter. The procyanidinmonomers include (+)catechin, (−)-epicatechin and their respectiveepimers (i.e., (−)-catechin and (+)-epicatechin). The oligomers whichhave been identified in cocoa extracts include the dimers through theoctadecamers. Cocoa procyanidins can also be obtained by syntheticmethods described in PCT/US98/21392 (published as WO 99/19319 on Apr.22, 1999) which is incorporated herein by reference. The oligomers maybe linear or branched. The linear 4 8 oligomers have the structure:

[0035] where x is an integer from 0-16. The branched oligomers have thestructure:

[0036] where a and b are independently integers from 0-15.

[0037] Structural variations to procyanidin oligomers may also occurwith the formation of a second interflavonoid bond by carbon-oxygenoxidative coupling to form A-type oligomers, as shown below (Porter etal. “Flavans and Proanthocyanidins” Chapter Two in “The Flavonoids”,Ed., J. B. Harborne, Chapman and Hall Ltd., London, 1988; Porter InMethods in Plant Biochemistry, Vol. I. Plant Phenolics. Dey andHarborne, Eds; Academic Press: San Diego, Calif., 1989). Due to thecomplexity of this conversion, A-type proanthocyanidins are not asfrequently encountered as the single-bonded oligomers:

[0038] beans, mixtures of purple and brown cocoa beans, or mixture ofslaty, purple, and brown cocoa beans. More preferably, the cocoa beansarc slaty and/or purple. Unfermented cocoa beans have a higher cocoapolyphenol content than fermented beans (Kim and Keeney J. Food Sci. 491090, 1984; Porter et al., “Cacao Procyanidins: Major Flavonoids andIdentification of Some Minor Metabolites” Phytochemistry, Vol. 30, No.5, 1657-1663, 1991).

[0039] The cocoa polyphenol content of cocoa beans or blends thereof ishigher when the beans have a fermentation factor of 275 or less. The“fermentation factor” is determined using a grading system forcharacterizing the fermentation of the cocoa beans. For example, slatybeans are designated 1, purple beans as 2, purple/brown beans as 3, andbrown beans as 4. The percentage of beans falling within each categoryis multiplied by the weighted number. Thus, the “fermentation factor”for a sample of 100% brown beans would be 100×4 or 400, whereas for a100% sample of purple beans it would be 100×2 or 200. A sample of 50%slaty beans and 50% purple beans would have a fermentation factor of 150[(50×1)+(50×20)].

[0040] Preferably, the unfermented or underfermented cocoa beans willcontain at least 1% by weight of total cocoa procyanidins based on theweight of the nonfat cocoa solids, preferably 3-7% by weight morepreferably 7-12% by weight, and most preferably 13-15%.

[0041] A method for preparing a cocoa mass suitable for extraction isdescribed in U.S. Pat. No. 5,554,645 (issued Sep. 10, 1996 to Romanczyket al.), the disclosure of which is herein incorporated by reference.Harvested cocoa pods were opened and the beans with pulp were removedfor freeze-drying. The pulp was manually removed from the freeze-driedmass and the beans were subjected to the following manipulations. Thefreeze-dried cocoa beans were first manually dehulled and ground to afine powdery mass with a TEKMAR Mill. The resultant mass was thendefatted overnight by Soxhlet extraction using redistilled hexane as thesolvent. Residual solvent was removed from the defatted mass by vacuumat ambient temperature.

[0042] Alternatively, the cocoa beans are sun dried under conditionswhich retard fermentation and the beans can be mechanically dried andthen dehulled.

[0043] Partially defatted or nonfat cocoa solids having a higher cocoapolyphenol content, i.e., a high cocoa procyanidin content, can beobtained by processing the cocoa bean or cocoa nib without the roastingstep and then milling the beans to chocolate liquor and recovering thepartially defatted cocoa solids, or by screw pressing the roasted beansor nibs to partially defatted cocoa solids without a milling step. Evenhigher levels can be achieved if underfermented cocoa beans are used inthis process. This method conserves the cocoa polyphenols because itomits the traditional roasting step. The method consists essentially ofthe steps of: (a) heating the cocoa beans to an internal beantemperature just sufficient to reduce the moisture content to about 3%by weight and loosen the cocoa shell, typically using a infra redheating apparatus for about 3 to 4 minutes; (b) winnowing the cocoa nibsfrom the cocoa shells; (c) screw pressing the cocoa nibs; and (d)recovering the cocoa butter and partially defatted cocoa solids whichcontain cocoa polyphenols including cocoa procyanidins. Optionally, thecocoa beans are cleaned prior to the heating step, e.g., in an airfluidized bed density separator. Preferably, the cocoa beans are heatedto an internal bean temperature of about 100° C. to about 110° C., morepreferably less than about 105° C. The winnowing can be carried out inan air fluidized bed density separator. The above process of heating thecocoa beans to reduce the moisture content and loosen the cocoa shell isdisclosed in U.S. Pat. No. 6,015,913 (issued Jan. 18, 2000 to KirkKealey) which is herein incorporated by reference.

[0044] The internal bean temperature (IBT) can be measured by filling aninsulated container such as a thermos bottle with beans (approximately80-100 beans). In order to maintain the temperature of the beans duringtransfer from the heating apparatus to the thermos, the insulatedcontainer is then appropriately sealed in order to maintain thetemperature of the sample therein. A thermometer is inserted into thebean filled insulated container and the temperature of the thermometeris equilibrated with respect to the beans in the thermos. Thetemperature reading is the IBT temperature of the beans. IBT can also beconsidered the equilibrium mass temperature of the beans.

[0045] In the context of this application, the following definitionsapply. As used herein, higher oligomers include the hexamers to thedecamers or higher and lower oligomers refer to procyanidins from thedimers up to and including the pentamers. As used herein, a cocoaextract is an extract containing an unspecified mixture of compoundswhich are extracted from ground cocoa beans, preferably dehulled beans,by slurrying defatted cocoa with an organic solvent and/or water andremoving the cocoa solids to yield a liquid. The liquid extract can bedried by conventional methods known to those skilled in the art.

[0046] The extraction process comprises the steps of slurrying thepartially defatted or nonfat cocoa solids with an appropriate solvent oraqueous solvent-water mixture to solubilize the cocoa polyphenols,centrifuging the slurry to separate the extracted cocoa solids from thesolvent phase, and removing the solvent(s). Preferably, the extractionprocess is a continuous process.

[0047] The solvent used can be any food grade organic solvent which willsolubilize the cocoa procyanidin monomers and oligomers present in thecocoa solids. Preferably, the solvent is chosen from the group,consisting of lower alcohols such as methanol, ethanol, and isopropanolor ethyl acetate and acetone. The solvents can be used in aqueoussolution or undiluted. Surprisingly, preferable dilutions vary withsolvent used.

[0048] Acidification of the solvent mixture to a pH of about 2 to about4 can either improve or reduce the efficiency of the procyanidinextraction depending upon the solvent used in the extraction procedure.The acid used in the reaction can be any food-grade, non-mineral acid.Preferred acids include formic acid, citric acid, phosphoric acid and,most preferably, acetic acid. Acids are not preferred if the extractionis carried out at a higher temperature.

[0049] The cocoa extracts can be detheobrominated and decaffeinated andthe methylxanthines can be recovered and purified. The preferred solventdiffers according to whether or not the methylxanthine is to bediscarded or recovered. If it is to be discarded, the ideal extractionsolvent is isopropanol at a low temperature. However, if themethylxanthine is to be recovered for purification, the ideal solvent isethanol and the extraction should be carried out at between 50-70° C.The optimum conditions for methylxanthine removal must be balancedagainst the optimum conditions for procyanidin extraction to provide aneffective simple process for the production of a methyl xanthine-freecocoa polyphenol extract.

[0050] The solvent extraction of the defatted cocoa solids can becarried out using either a batch process or a continuous counter-currentprocess. In the batch process a large solvent to solids ratio isrequired to extract the procyanidins from the cocoa solids.Additionally, several consecutive washes are usually required to extractall of the available procyanidins. The preferred method of extraction isa continuous counter-current extraction. In this process the efficiencyof extraction and the cost-effectiveness of the process is optimized,allowing it to be scaled up for manufacturing purposes. Use of acontinuous counter-current flow of solvent through the cocoa solidsallows the solvent to solids ratio to be much lower since the solventcan be continuously recycled. Additionally, due to the continuous flowof the solvent through the cocoa solids, it is not necessary to performdual solvent extractions or to wash the solids with an aqueous solventto ensure that all the procyanidins are extracted from the cocoa solids.

[0051] As will be shown hereafter, the efficacy of the procyanidinextraction varies significantly depending upon which solvent is used.Furthermore, it has been surprisingly found that while some solventswill preferentially extract the higher oligomers, other solvents willpreferentially extract the monomers and lower oligomers. The type ofsolvent used, whether or not the solvent is an aqueous solution, thetemperature at which the extraction is carried out, and other factorscan affect the selective extraction of high or low molecular weightprocyanidin oligomers. By sequentially carrying out the extraction ofthe same cocoa powder with two separate solvent systems, one known toextract monomer and the lower oligomers, and the other known to extractthe higher oligomers, it is possible to obtain separate procyanidinfractions from the same extraction procedure, without following theextraction with complicated separation methods. Thus, a furtherembodiment of the invention is a process by which oligomers can beselectively extracted from cocoa solids in a large-scale, single stepprocess which avoids the costly and complex process ofchromatographically separating the oligomers.

Test Procedures Control Extract

[0052] In the examples which follow all solvent extractions werecompared to control extracts which were prepared by extractingunfermented, freeze-dried, defatted, cocoa beans prepared using theprocedures described in Part A of Example 1. The solvent used was amixture of 70% acetone, 29.5% water, and 0.5% acetic acid. All aqueoussolutions are expressed as % (v/v). Ten grams of defatted cocoa mass wasslurried with 100/mL of this solvent mixture and sonicated for 5-10 min.at 50° C. The slurry was centrifuged for 15 min. at 4° C. at 3000×G andthe supernatant was passed through glass wool. The yields of crudeprocyanidins ranged from 15-20%.

[0053] Separation and Quantification of The Procyanidin OligomersPresent in the Cocoa Extracts

[0054] In the examples which follow, the analytical methods describedbelow were used to separate and quantify, by degree of polymerization,the procyanidin composition of the cocoa extracts. The method, describedbelow, of identifying procyanidin oligomers in the cocoa extract isbased upon work reported in Hammerstone, J. F. et al, “Identification ofProcyanidins in Cocoa (Theobroma cacao) and Chocolate UsingHigh-Performance Liquid Chromatography/Mass Spectrometry”, J. Ag. FoodChem.; 1999; 47 (10) 490-496. The analytical methods described belowwere used in a qualitative study of a broad range of food and beveragesamples reported to contain various types of proanthocyanidins, asreported in Lazarus, S. A., et al. “High-performance LiquidChromatography/Mass Spectrometry Analysis of Proanthocyanidins in Foodsand Beverages”, J. Ag. Food Chem.; 1999; 47 (9); 3693-3701. The methodof Lazarus et al. (1999) reported analysis using fluorescence detectionbecause of higher selectivity and sensitivity.

[0055] The samples were analyzed using the analytical method reported inAdamson, G. E. et al., “HPLC Method for the Quantification ofProcyanidins in Cocoa and Chocolate Samples and Correlation to TotalAntioxidant Capacity”, J. Ag. Food Chem.; 1999; 47 (10) 4184-4188.Samples were then compared directly to the control extract to accuratelydetermine the levels of procyanidins monomers and oligomers incomparison to the monomers and oligomers in the control extract.

[0056] The total amount of procyanidins in an extract can also bedetermined using the method reported in Adamson et al. However, insteadof comparing sample procyanidin levels with those of the controlextract, the samples are compared with composite standard stocksolutions of each of the monomers and oligomers, allowing the absoluteamount of each procyanidin present in the extract to be calculated.Composite standard stock solutions and calibration curves were generatedfor the cocoa procyanidin monomers and oligomers by the method reportedin Adamson at al.

EXAMPLES Example 1 Cocoa Source and Preparation of Cocoa PowderContaining Cocoa Polyphenols Part A—Preparation From Freeze-Dried CocoaBeans

[0057] Methods for preparing a defatted cocoa mass are described in U.S.Pat. No. 5,554,645 (issued to Romanczyk Sep. 10, 1996) which is hereinincorporated by reference. Unless otherwise stated, the cocoa beans usedwere unfermented and were processed in the following manner. Harvestedcocoa pods were opened and the beans with pulp were removed forfreeze-drying. The pulp was manually removed from the freeze-dried massand the beans were subjected to the following manipulations. Thefreeze-dried cocoa beans were first manually dehulled and ground to afine powdery mass with a TEKMAR Mill. The resultant mass was thendefatted overnight by Soxhlet extraction using redistilled hexane as thesolvent. Residual solvent was removed from the defatted mass by vacuumat ambient temperature.

Part B—Preparation From Roasted Cocoa Beans

[0058] The cocoa powder was prepared by a conventional method whichcomprises the steps of roasting the cocoa beans to an internal beantemperature of 95° C. to 160° C., winnowing the cocoa nibs from theroasted cocoa beans, milling the roasted cocoa nibs into chocolateliquor, pressing the liquor to extract the cocoa butter, and recoveringthe cocoa butter and partially defatted cocoa solids. The cocoa solidscan be further defatted using hexane as a solvent, as previouslydescribed.

Part C—Preparation from Non-Roasted Cocoa Beans

[0059] Partially defatted cocoa solids having a high cocoa polyphenolcontent, i.e., a high cocoa procyanidin content, were obtained byprocessing the sun-dried, unfermented cocoa beans to cocoa solidswithout a bean or nib roasting step and, if desired, without the step ofmilling the beans to chocolate liquor, i.e., the beans can be screwpressed to provide cocoa butter and partially defatted cocoa solids. Thecocoa polyphenols were conserved because the traditional roasting stepwas omitted. The method includes the steps of: (a) heating the cocoabeans to an internal bean temperature just sufficient to reduce themoisture content to about 3% by weight and loosen the cocoa shell; (b)winnowing the cocoa nibs from the cocoa shells; (c) screw pressing thecocoa nibs; and (d) recovering the cocoa butter and partially defattedcocoa solids which contain cocoa polyphenols including cocoaprocyanidins. Typically, the heating is carried out in an infra redheating apparatus for about 3 to 4 minutes. Optionally, the cocoa beansare cleaned prior to the heating step, e.g., in an air fluidized beddensity separator. Preferably, the cocoa beans were heated to aninternal bean temperature of about 100° C. to about 110° C., morepreferably less than about 105° C. The winnowing can be carried out inan air fluidized bed density separator. The above process of heating thecocoa beans to reduce the moisture content and loosen the cocoa shell isdisclosed in U.S. Pat. No. 6,015,913 (issued Jan. 18, 2000) which isherein incorporated by reference.

Example 2 Improved Extraction Processes

[0060] In the following series of experiments, the solvents andconditions used for the extractions were varied and compared in order toassess the efficiency of the different extraction processes. In additionto comparing the efficacy of various solvents, the effects of varyingthe extraction temperature, pH, and the ratio of water to solvent usedwere also compared.

Part A—Effect of Solvents at 50° C.

[0061] Extracts were prepared using cocoa solids prepared fromunfermented, freeze-dried, defatted, cocoa beans as described in Part Aof Example 1. Ten grams of the defatted cocoa mass were slurried with100 mL of a solvent for 5-10 min. at 50° C. The solvents used wereethanol, methanol, isopropanol, and acetone. The solvent to waterpercentage was varied between 50-100%. The slurry was centrifuged for 5min. at ambient temperature C at 3000×G and the supernatant passedthrough a 0.45 micron filter. The amount of procyanidins extracted andthe oligomeric profile of the cocoa extract were determined using theanalytical methods described above. The quantity of each oligomerdetected was expressed as a percentage of the amount of the sameoligomer extracted using the control extract described above.

Part B—Effect of pH

[0062] The effect of the addition of 0.5% acetic acid to the solventswas also studied. The extractions were carried out at temperatures 0-70C. These results are shown in Tables 7-8. Surprisingly, the presence orabsence of 0.5% acetic acid in the solvent or aqueous solvent mixturehad a variable effect on the extraction efficacy depending on thesolvent being used and the solvent concentration.

Isopropanol and Isopropanol/Water

[0063] As can be seen from a comparison of the data in Tables 1 & 2, forisopropanol (IPA), the addition of acid resulted in an overall increasein oligomeric extraction at all solvent dilutions. However, the effectof adding acid to the 80% isopropanol doubled the amount of loweroligomers extracted, while leaving the amount of higher oligomersextracted largely unaltered.

Methanol and Methanol/Water

[0064] The addition of acid to the aqueous methanol solvents resulted inan increase in oligomeric yield. The 100% methanol, however, wasunaffected by the presence of acid. This data is shown in Tables 3 and4.

Acetone and Acetone/Water

[0065] The addition of acid to the acetone enhanced the extraction ofmonomers and dimers through heptamers, but had no effect on theextraction of the higher oligomers. The results are shown in Tables 5and 6.

Ethanol and Ethanol/Water

[0066] The addition of acid to the aqueous ethanol solvents had verylittle effect on the extraction of monomers and oligomers up to theheptamers, but it enhanced the extraction of the higher oligomers. Theextraction of the heptamers and higher oligomers was significantly moreeffective when there was no acid present in the 100% ethanol. This datais shown in Tables 7 & 8.

Part C—Effect of Water

[0067] The amount of water used in combination with the methanol,ethanol, isopropanol, and acetone also had variable effects on theefficacy of the extraction of the cocoa procyanidins depending on theorganic solvent used.

Isopropanol

[0068] There was a pronounced decline in procyanidin extraction withincrease in oligomeric size for all isopropanol (IPA) dilutions.However, 80% IPA was the least effective aqueous solvent. The 50, 60 &70% aqueous IPAs were the most effective for all oligomers apart fromthe decamers, for which 100% IPA was the most effective. It should benoted that for the monomer and lower oligomers (up to the pentamer), the50-70% IPAs were more than twice as effective as the 80 and 100% IPAs.The data are shown in Tables 1 and 2.

Methanol and Ethanol

[0069] The data in Tables 3, 4, 7 & 8 show that when methanol or ethanolwas used, the 70 and 80% aqueous solvents gave the best extractions oflower oligomers, while 100% methanol and ethanol were the most effectivefor the higher oligomers. For oligomers higher than the pentamer, thelower concentrations of solvent became less effective with increasingoligomeric size, while the 100% methanol and ethanol were moreeffective.

Acetone

[0070] The extraction efficiency of the lower solvent concentrations (50and 60%) increased with oligomeric size, while the extraction efficiencyof the higher solvent concentrations (80 and 100%) decreased witholigomer size. The data are shown in Tables 5 and 6.

[0071] One skilled in the art will recognize that the choice of whetherto use 100% of solvent or an aqueous-solvent mixture will depend on theoligomeric profile of the procyanidins desired in the cocoa extract.

Part D—The Effect of Temperature

[0072] Experiments were carried out to determine the effect oftemperature on the extraction of cocoa procyanidins using methanol,ethanol, isopropanol, and acetone. The extractions were carried out asdescribed above, except that they were carried out at 0° C., 20° C., 50°C. and 70° C. except for methanol which has a boiling point of 64° C.When 100% isopropanol was used as the solvent, the efficiency ofextraction of all the procyanidin oligomers increased dramatically asthe extraction temperature was increased (see FIG. 1b). When 100%ethanol was used as the solvent, the extraction efficiency for alloligomers increased until 50° C., (see FIG. 1A). When 100% methanol wasused the effect of temperature varied with oligomeric size. For example,the yield of the monomers was unaffected by temperature; the yield ofthe dimers to the heptamers increased as the temperature was increasedfrom 0° C. to 20° C.; and yield of the higher oligomers (octamers todecamers) increased substantially when the temperature was increased to20° C., but above 20° C. the yield decreased (see FIG. 1c). The dataillustrated in FIG. 1a, 1 b and 1 c are shown in Tables 9-11.

Example 3 Comparison of Solvent Effectiveness in Extracting the CocoaProcyanidins

[0073] For a general comparison of solvent effectiveness see FIG. 2. Foreach solvent, the dilution which gave the best overall procyanidin yieldwas selected. As can be seen, the differences in the yields of themonomers and oligomers up to the hexamers did not vary greatly betweenthe solvents. However, for the higher oligomers it became clear thatsome solvents were preferentially extracting the lower oligomers, whileothers were preferentially extracting the higher oligomers. For example,isopropanol was an extremely effective solvent for the monomer and loweroligomers, but its efficacy dropped off dramatically for the higheroligomers. Conversely, ethanol was the least effective solvent forextracting the monomer and lower oligomers, but was highly effective forextract the higher oligomers. Overall, the best solvent mixture for theextraction of the cocoa procyanidins was a mixture of 50% acetone, 49.5%deionized water, and 0.5% acetic acid, and this extraction efficiencyincreased with oligomeric size in comparison with all other solventsystems tested, including the control system.

Example 4 The Effect of Fermentation and Subsequent Processing of theCocoa Beans on the Extraction of Cocoa Procyanidins.

[0074] This example studies the effect of fermentation on theprocyanidin yield by comparing the differences in procyanidin recoverylevels from the following groups of cocoa beans. The first group ofcocoa beans were not fermented and were freeze-dried and groundaccording to the method of Part A of Example 1. The second group ofcocoa beans were removed from the pod and left in a pile for a period oftime sufficient for fermentation to occur, after which they wereprocessed to cocoa solids using the same method as that used for theunfermented cocoa beans. The amount of time required for normalfermentation to occur varies according to the size of the mass of cocoapods and the frequency with which the pods are turned. It takes aboutthree days for beans to be lightly fermented, between five and sevendays for full fermentation to take place, and beans will beover-fermented after eight days. The third group of cocoa beans were notfermented and were not roasted. Rather, they were removed from the pod,sun-dried, and processed according to the method disclosed in the U.S.Pat. No. 6.015,913 (to Kealey et al.). The process involves heating thecocoa beans only for a time and temperature sufficient to reduce themoisture content and loosen the cocoa shells so that the shells can beseparated from the nibs in the winnowing step. The beans weresubsequently reduced to a powder by the method of Example 1. The cocoaprocyanidins were extracted using the “control” extraction method wherethe solvent was a mixture of 70% acetone, 29.5% water and 0.5% aceticacid. The amount of each of the procyanidin monomers and oligomerspresent in each extract was determined using the analytical procedurepreviously described. As shown in FIG. 3, extracts from fermented beanscontained between 75 to 100% less cocoa procyanidins than the extractprepared from unfermented beans. The relative loss of procyanidinsincreased with oligomeric size. Heating the beans to remove the shellsalso resulted in extract with reduced cocoa procyanidin levels. Thecocoa extract from the heated cocoa beans contained levels ofprocyanidin oligomers which ranged between 10-50% of those found in thecocoa extract from the unfermented cocoa beans.

[0075] The results show that the fermentation and subsequent processingof the cocoa beans have a more dramatic effect on the procyanidincontent of the extract than the choice of solvent(s).

[0076] The above experiment was repeated using the same groups of beans,but altering the method of extraction by using boiling water as thesolvent. As can be seen in FIG. 4, the use of boiling water as thesolvent drastically reduced the overall yield of cocoa procyanidins. Theloss increased with oligomeric size such that the higher oligomers werenot extracted using hot water.

Example 5

[0077] This example is a comparative example which repeats theextraction process described in a group of Japanese applications andpatents owned by a Meiji Seika Kaisha Ltd., where hot aqueous ethanol isthe preferred solvent. See JP 9206026, published Aug. 12, 1997; JP7274894, published Oct. 24, 1995; JP 9224606, published Sep. 2, 1997. Inthese publications, it is not disclosed if the beans were fermented androasted. Presumably, the cocoa beans were prepared by the “traditional”method of fermentation followed by roasting and winnowing. As shownabove, fermentation and roasting deplete the available supplies ofprocyanidins in the cocoa bean.

[0078] Experiments were carried out to compare the amount ofprocyanidins and the oligomeric profile of the extract obtained when anextraction was carried out using (a) fermented, (b) underfermented and(c) unfermented, non-roasted cocoa beans processed according to theprocedure of U.S. Pat. No. 6,015,913 (Kealey et al.). Cocoa solids wereprepared from the three groups of beans by the methods described in PartA of Example 1. Two extracts were made from each group. The firstextract was prepared at 50° C. using the standard solvent mixture ofacetone/water/acetic acid (70%/29.5%/0.5%) as the solvent. The secondextract was prepared at 80° C. using a mixture of 80% ethanol and 20%water as the solvent. The amount of each oligomer present in the aqueousethanol extract was reported as a percentage of the amount oligomerpresent in the standard acetone/water/acetic acid extract.

[0079] The results are shown in Table 14. The yield of procyanidins wasdramatically reduced when the extraction was carried out using fermentedbeans. Furthermore, there were virtually no higher oligomers in theextract from the fermented beans. The yield of higher oligomers washighest when the extraction was carried out using the same solvent butusing unfermented beans. The use of 100% ethanol as the solvent resultedin extremely high yields of the nonamers and decamers (103% for thenonamer and 110% for the decamer compared to the 100% for the standardsolvent). The extraction with 80% ethanol gave much lower yields (37%for the nonamer, 18% for the decamer) compared to 100% for the standardsolvent.

Example 6 Extraction of Cocoa Procyanidins From Cocoa Powder Using aCounter Current Extraction Method

[0080] In a continuous counter-current extraction process, an organicsolvent or an aqueous organic solvent passes through an extractionsystem comprising a plurality of cells containing the partially defattedcocoa solids. The organic solvent or aqueous-organic solvent mixtureenters the extraction system at a cell containing the most extractedbatch of cocoa solids, passes through progressively fresher batches ofcocoa powder contained in successive cells, and is finally drawn offfrom the cell containing the freshest batch of cocoa solids. Thus, thesolvent and the solid to be extracted are moving through the extractionsystem counter-current to one another. The temperature of the solventmixture entering the cell containing the most extracted cocoa materialmay be from about 20° C. to about 100° C., preferably from 50° C. to 95°C. at atmospheric pressure, or higher if the extraction takes placeunder pressure. The solvent should be in the liquid, as opposed to thegaseous phase, therefore the reaction temperature must either be belowthe boiling point of the solvent or the reaction must be carried out ina pressurized vessel. Since experimental data generated in the batchprocesses suggests that the extraction process is more effective athigher temperatures, it may be preferable to carry out the countercurrent extraction at a higher pressure. The number of cells and cycletime are chosen to give the maximum yields of extracted procyanidinsfrom the cocoa solids. The procyanidin content of the extract isconcentrated by recycling the extract in the continuous counter-currentsystem. In such a system the extract passes continuously through thecolumns in series. Periodically, the cell containing the most extractedcocoa solids is removed from the system and one containing fresh cocoasolids is added. The optimum number of cells in series which are used,and the length of each cycle, will vary with particle size of the cocoasolids and extraction parameters such as temperature and pressure.Methods of determining the optimum conditions are well known to thoseskilled in the art. See “Unit Operations of Chemical Engineering” 3^(rd)Edition, Chapter 7, Eds. McCabe, W., and Smith J., McGraw Hill,incorporated herein by reference.

Example 7 Selective Extraction of Procyanidin Oligomers According toTheir Size

[0081] This example demonstrates how extracts containing predominantlyhigh molecular weight cocoa procyanidin oligomers or predominantly lowmolecular weight oligomers can be obtained by the selective use ofsolvents which preferentially solubilize certain oligomers.

[0082] One gram of cocoa powder, obtained from unfermented cocoa beansby the methods described in Example 1, was extracted three times with100% ethyl acetate at 50° C. The extracts were combined and concentratedby rotary evaporation under vacuum until dry. The dry extract wasreconstituted with 10 mls of the standard acetone/water/acetic acid(70%/29.5%/0.5%) solvent and purified and analyzed for oligomericcontent as described previously. The ethyl acetate-extracted cocoasolids were then dried in a vacuum oven overnight to remove any residualsolvent. The dried solids were extracted with the standardacetone/water/acetic acid solvent (70%/29.5%/0.5%) at 50° C., purified,and then analyzed for oligomeric content as described previously. Theoligomeric content of both extracts was determined as a percentage ofthe oligomeric content of an extract prepared from unfermented cocoabeans using the standard acetone/water /acetic acid solvent(70%/29.5%/0.5%) at 50° C. As can be seen in Table 13 and FIG. 7, theuse of ethyl acetate as the solvent resulted in the extraction of onlythe monomer and lower oligomers, leaving all the higher oligomers in theextracted cocoa solids. The higher oligomers were then efficientlyextracted using the standard acetone/water/acetic acid solvent(70%/29.5%/.05%) or 100% ethanol.

Example 8 Detheobromination of the Cocoa Extract

[0083] In these experiments, various solvents were used at a range oftemperatures to extract cocoa procyanidins from cocoa solids. Theextracts were then analyzed to quantify the amount of caffeine andtheobromine present in the cocoa extracts. The solvents used wereisopropanol, ethanol and methanol. The extractions were carried out at0°, 20°, 50° and 70° C. using the method described in Example 1.

[0084] The results are shown in Table 12. The yields of theobromine werethe greatest when ethanol was used as the solvent and the least whenisopropanol was used as the solvent. The yields increased with increasedtemperature. Thus, the results show that theobromine is most soluble inethanol and least soluble in isopropanol, and that solubility increaseswith temperature.

[0085] To achieve minimum theobromine extraction, and thereforefacilitate its removal, the optimum extraction will be extraction withisopropanol carried out at the lowest temperature which results in theextraction of the procyanidins. The theobromine will then beprecipitated out of the extract by chilling the extract overnight atabout 0°-4° C. and removed by filtering the extract.

[0086] To recover the theobromine from cocoa beans, the ideal extractionwill be with ethanol at between 50°-70° C. Subsequent to the extraction,the ethanol extract will be chilled to between 0-5° C., which shouldresult in the precipitation of the theobromine. The theobromine isseparated by filtration, by centrifugation in a contiguous centrifuge,or by adsorption onto a neutral adsorbent followed by elution.

[0087] Preferably, the theobromine is collected as a crude solid bypassing the theobromine-laden solvent vapor through a system of chilledplates within closed cabinets (as shown schematically in FIG. 6). Thesolvent is then be purged from the system and recovered for further use.Meanwhile, the crude theobromine is scraped off the chilled plates, anddissolved in an aqueous solution. Purified, pharmaceutical gradetheobromine is obtained by recrystallizing the theobromine, drying thecrystals, and redissolving them in de-ionized double-distilled water.

[0088] Alternatively, the methylxanthines can be removed from theextract using gel permeation chromatography, as described in U.S. Pat.No. 5,554,645, (issued to Romanczyk et al., Sep. 10, 1996) Briefly, thepartial purification process was carried out using liquid chromatographyon Sephadex LH 20 (28×2.5 cm). Separations were aided by a step gradientinto deionized water. The initial gradient composition started with 15%methanol in deionized water, which was followed step-wise every 30minutes with 25% methanol in deionized water, 35% methanol in deionizedwater, 70% methanol in deionized water, and finally 100% methanol. Theeffluent following the elution of caffeine and theobromine was collectedas a single fraction, which represents a xanthine alkaloid freesub-fraction of the original extract.

[0089] The methylxanthines can also be removed from the cocoa extract byadsorbing them onto a solid adsorbent, after which the extract,substantially free of caffeine and theobromine, is washed through theadsorbent. Various solid adsorbents can be used in the process, such aspolymeric resins and activated carbon. Preferably, the adsorbent issubstantially neutral in water: and example of a neutral resin adsorbentis semi-calcinated resin XE-340, manufactured by Rohm & Haas; neutralactivated carbon ca be obtained by either by acid washing of thermallyactivated carbon followed by rinsing with water to neutrality, or byneutralization of acid-activated carbon with an aqueous alkali followedby rinsing with water to neutrality. TABLE 1 Average % Recovery UsingIsopropanol or Aqueous Isopropanol 0.5% Acid at 50° C. % Isopropanolmonomer dimer trimer tetramer pentamer hexamer heptamer octamer nonamerdecamer 50% 94.92 91.17 90.37 90.53 84.23 78.98 69.86 60.42 43.83 35.7960% 97.26 93.03 91.17 89.46 80.58 72.75 59.97 50.09 35.11 26.49 70%94.96 90.52 86.12 78.97 64.32 50.80 35.59 25.53 14.56 8.60 80% 94.7086.16 73.96 57.96 38.61 24.02 12.71 6.66 0.00 0.00 100%  58.45 50.8950.12 49.49 46.67 44.33 40.38 38.49 34.85 29.12

[0090] TABLE 2 Average Percentage Recovery Using Isopropanol or AqueousIsopropanol without Acid at 50° C. % Isopropanol monomer dimer trimertetramer pentamer hexamer heptamer octamer nonamer decamer 50% 93.2588.96 86.61 84.58 82.82 75.01 66.54 60.77 50.05 18.18 60% 92.59 87.9084.57 80.67 79.46 63.73 55.08 48.00 36.44 9.09 70% 93.32 90.43 83.0973.31 65.00 45.45 30.11 24.13 14.15 1.52 80% 40.52 35.71 25.75 20.3817.77 14.16 11.93 11.73 8.88 1.61 100%  42.14 38.95 37.56 36.24 35.8132.10 32.05 30.75 28.22 22.14

[0091] TABLE 3 Average Percentage Recovery Using Methanol or AqueousMethanol with 0.5% Acid at 50° C. % Methanol monomer dimer trimertetramer pentamer hexamer heptamer octamer nonamer decamer 50% 86.9885.48 80.24 74.98 72.26 58.27 55.21 50.00 39.88 0.00 60% 91.60 89.6685.22 81.26 77.00 66.19 56.50 48.93 31.90 0.00 70% 92.46 95.23 95.5797.15 90.90 84.33 72.67 60.82 41.82 28.95 80% 88.53 94.10 92.86 92.4385.80 78.57 66.00 54.12 36.36 21.05 100%  92.40 90.73 90.76 92.06 92.8286.94 80.34 77.63 61.13 43.86

[0092] TABLE 4 Average Pecentage Recovery Using Methanol without Acid at50° C. % Methanol monomer dimer trimer tetramer pentamer hexamerheptamer octamer nonamer decamer 50% 79.50 74.32 69.48 63.73 56.62 46.9235.86 28.09 17.02 8.33 60% 81.04 76.33 72.66 67.65 62.15 52.56 39.8032.58 20.21 12.50 70% 87.07 82.60 76.19 39.18 60.85 51.18 42.18 36.7135.48 18.75 80% 89.49 84.94 77.82 70.78 62.09 52.35 42.18 34.18 29.0312.50 100%  84.94 76.41 77.73 78.06 76.62 76.41 69.74 71.91 62.77 66.67

[0093] TABLE 5 Average Percentage Recovery Using Acetone with 0.5% Acidat 50° C. % Acetone monomer dimer trimer tetramer pentamer hexamerheptamer octamer nonamer decamer 50% 95.93 93.96 93.97 95.69 99.64107.73 114.39 112.33 126.81 121.05 60% 95.28 94.81 94.84 95.78 97.65100.78 104.29 103.62 111.35 111.40 80% 97.14 95.21 95.56 94.08 92.5791.95 94.11 76.32 82.86 66.91 100%  77.30 73.43 74.55 77.56 76.03 74.0563.03 54.02 13.04 13.16

[0094] TABLE 6 Average Percentage Recovery Using Acetone Without Acid at50° C. % Acetone monomer dimer trimer tetramer pentamer hexamer heptameroctamer nonamer decamer 50% 89.36 84.69 86.20 85.61 86.67 94.77 94.17108.94 108.21 140.59 60% 98.18 115.95 75.54 98.67 100.16 102.95 102.95105.39 100.94 131.95 80% 103.54 103.34 102.89 100.64 94.97 93.21 80.2380.56 68.06 24.42 100%  72.07 68.20 69.57 73.20 73.88 78.36 71.70 68.660.00 0.00

[0095] TABLE 7 Average Percentage Recovery Using Ethanol with 0.5% Acidat 50° C. % ETHANOL monomer dimer trimer tetramer pentamer hexamerheptamer octamer nonamer decamer 50% 86.02 83.17 80.77 79.85 77.00 69.9466.73 52.51 37.80 19.09 60% 88.12 84.02 81.00 77.88 75.92 68.33 61.8344.00 32.21 15.76 70% 87.97 82.66 78.61 74.24 68.21 58.39 48.77 32.9918.87 3.33 80% 91.49 82.90 75.51 67.29 57.72 45.53 36.21 24.08 13.340.00 100%  81.48 75.53 75.15 75.76 76.69 76.10 78.53 71.12 68.92 63.03

[0096] TABLE 8 Average Percentage Recovery Using Ethanol without 0.5%Acid at 50° C. % ETHANOL monomer dimer trimer tetramer pentamer hexamerheptamer octamer nonamer decamer 50% 84.43 82.32 79.33 78.40 76.55 67.2153.33 39.88 29.07 3.33 60% 87.99 86.04 82.58 81.69 78.17 66.74 51.1136.90 23.26 6.67 70% 87.96 83.40 78.00 74.01 67.99 55.58 41.11 28.5717.44 6.67 80% 91.85 86.62 83.11 81.00 71.02 63.13 53.08 43.75 37.3618.33 100%  89.44 81.87 83.24 87.94 85.76 90.00 95.29 97.75 102.91109.86

[0097] TABLE 9 Effect of Temperature on Percentage of Cocoa ProcyanidinMonomers and Oligomers Extracted using 100% Ispropanol ° C. monomerdimer trimer tetramer pentamer hexamer heptamer octamer nonamer decamer0 2.44 1.94 1.71 1.47 1.12 0.88 0.68 0.46 0.00 0.00 20 5.85 4.97 4.544.11 3.55 3.06 2.63 2.39 0.93 1.03 50 38.21 36.43 36.04 35.27 33.5731.62 30.56 30.44 25.27 25.89 70 72.76 69.92 68.99 65.72 62.41 59.3057.32 60.45 54.34 61.33

[0098] TABLE 10 Effect of Temperature on Percentage of Cocoa ProcyanidinMonomers and Oligomers Extracted using 100% Methanol ° C. monomer dimertrimer tetramer pentamer hexamer heptamer octamer nonamer decamer 091.22 89.43 85.98 83.10 79.01 76.36 69.78 68.47 58.15 37.72 20 92.2095.87 93.65 92.94 88.56 91.54 87.13 91.54 84.38 87.35 50 90.47 92.4191.91 90.22 88.33 87.46 86.85 88.67 82.52 80.69

[0099] TABLE 11 Effect of Temperature on Percentage of Cocoa ProcyanidinMonomers and Oligomers Extracted using 100% Ethanol ° C. monomer dimertrimer tetramer pentamer hexamer heptamer octamer nonamer decamer 067.66 68.17 67.84 67.49 63.93 62.28 58.98 53.72 49.69 46.06 20 73.2772.88 73.03 72.90 70.61 69.83 64.53 63.43 59.68 53.85 50 89.12 88.2289.39 91.22 88.84 86.35 86.26 86.87 73.57 80.90 70 90.52 88.21 89.1791.32 88.97 87.93 87.06 87.06 82.82 79.31

[0100] TABLE 12 Comparison of Oligomeric Yields Obtained by DualExtraction with Ethyl Acetate followed by Acetone/Water/Acetic Acidmonomer dimer trimer tetramer pentamer hexamer heptamer octamer nonamerdecamer Acetone/water/acetic acid extract from 100 100 100 100 100 100100 100 100 100 unfermented beans Ethyl acetate extract from unfermented47.77 34.94 25.77 17.63 9.98 5.28 3.43 0 0 0 beans Acetone/water/aceticacid extract of 66.47 72.81 78.5 83.45 89.05 92.96 96.08 97.87 100 100ethyl acetate-extracted cocoa solids

[0101] TABLE 13 Yields of Caffeine and Theobromine Extracted from CocoaBeans using Various Solvents at Various Temperatures. Solvents/Temp.Theobromine mg/g/ Caffeine mg/g isopropanol @ 0 C. 0 0 isopropanol @ 20C. Trace Trace isopropanol @ 50 C. 1.7 Trace isopropanol @ 70 C. 3.50.15 methanol @ 0 C. 1.17 Trace methanol @ 20 C. 1.56 0.16 methanol @ 50C. 1.88 0.19 ethanol @ 0 C. 1.16 Trace ethanol @ 20 C. 3.58 0.16 ethanol@ 50 C. 4.45 0.18 ethanol @ 70 C. 3.73 0.16

[0102] TABLE 14 A Comparison of Oligomeric Yields from Extracts Obtainedfrom Fermented and Unfermented Cocoa Beans using 80% Ethanol at 80 °C.monomer dimer trimer tetramer pentamer hexamer heptamer octamer nonamerdecamer powder from unfermented beans 93.27 105.99 99.58 95.26 81.0264.44 46.03 30 18.8 12.5 powder from fermented beans 64 56.41 50 36.8433.33 33.33 0 0 0 0

What is claimed is:
 1. An improved method for extracting cocoapolyphenols including cocoa procyanidins from partially or fullydefatted cocoa solids, prepared from cocoa beans that have not beenroasted, which method comprises a single extraction, at atmosphericpressure or under pressure, with a solvent selected from the groupconsisting of an organic solvent capable of solubilizing the cocoapolyphenols, or aqueous mixtures thereof, to which solvent oraqueous-solvent mixture is added an edible acid in an amount sufficientto lower the pH to about 2 to about
 4. 2. The method of claim 1, whereinthe cocoa solids are prepared from unfermented or underfermented cocoabeans and wherein the organic solvent is selected from the groupconsisting of methanol, ethanol, isopropanol, acetone, methyl acetate,or ethyl acetate and wherein the aqueous solvent mixture contains up toabout 50% water by volume.
 3. The method of claim 2, wherein theaqueous-solvent mixture is a mixture of isopropanol and about 50-70%water.
 4. The method of claim 2, wherein the aqueous-solvent mixture isa mixture of isopropanol and about 20% water.
 5. The method of claim 4,whereby the monomers and oligomers up to and including hexamers arepreferentially extracted.
 6. The method of claim 2, wherein theaqueous-solvent mixture is a mixture of acetone and about 80% to about100% water.
 7. The method of claim 2, wherein the aqueous-solventmixture is a mixture of acetone and about 50% to about 70% water.
 8. Themethod of claim 2, wherein the organic solvent is ethanol.
 9. The methodof claim 8, whereby the extraction of cocoa procyanidin oligomers higherthan the heptamers is enhanced.
 10. A method of extracting cocoapolyphenols including cocoa procyanidin monomers and oligomers frompartially defatted or fully defatted cocoa solids, prepared from cocoabeans that have not been roasted, comprises the step of extracting thecocoa solids, at atmospheric or under pressure, with a non-acidifiedorganic solvent capable of solubilizing cocoa polyphenols ornon-acidified aqueous mixtures thereof.
 11. The method of claim 10,wherein the cocoa beans are unfermented or underfermented cocoa beans.12. The method of claim 2 or 11, wherein the cocoa beans have afermentation factor of 275 or less.
 13. The method of claim 2 or 11wherein the cocoa beans are slaty, purple, or purple brown cocoa beans,or mixtures of slaty and purple cocoa beans, purple and brown cocoabeans, or slaty, purple and brown cocoa beans.
 14. The method of claim 2or 11, wherein the unfermented or underfermented cocoa beans contain atleast about 1% up to about 15% by weight of total cocoa procyanidins pergram of defatted cocoa solids.
 15. The method of claim 14, wherein theweight of the total cocoa procyanidins is about 4-7%.
 16. The method ofclaim 10, wherein the organic solvent is methanol, ethanol, isopropanol,acetone, methyl acetate, or ethyl acetate and wherein the aqueousmixture contains up to 50% water.
 17. The method of claim 10, whereinthe solvent is about 50 to about 70% acetone or about 95 -100%.
 18. Themethod of claim 1 or 10, wherein the extraction is carried out at atemperature of from 0° C. up to the boiling point of the solvent oraqueous solvent used.
 19. The method of claim 10, wherein the solvent ismethanol and wherein the extraction is carried out at room temperature,wherein the solvent is ethanol and the extraction is carried out atabout 20° to about 50° C., or wherein the solvent is isopropanol and theextraction is carried out at about 70° C.
 20. The method of claim 10,wherein the solvent is an aqueous acetone mixture and wherein theextraction is carried out at about 50° C. up to the boiling point of themixture at the extraction pressure used.
 21. A method for selectivelyextracting low molecular weight cocoa procyanidin oligomers frompartially or fully defatted cocoa solids, prepared from cocoa beans thathave not been roasted, which method comprises the step of extracting, atatmospheric pressure or under pressure, the cocoa solids with methylacetate or ethyl acetate.
 22. The method of claim 21, wherein the cocoabeans are unfermented or underfermented cocoa beans and wherein theextraction is carried out at about 20° C. to about 50° C.
 23. The methodof claim 22, wherein the lower oligomers consist essentially of themonomers, dimers, and trimers.
 24. A cocoa extract prepared by theprocess of claim
 22. 25. A cocoa extract consisting essentially ofprocyanidin monomers, dimers, and trimers.
 26. A method for selectivelyextracting higher molecular weight cocoa procyanidin oligomers frompartially defatted or fully defatted cocoa solids, prepared from cocoabeans that have not been roasted, which method comprises the steps of(a) extracting, at atmospheric pressure or under pressure, the cocoasolids with ethyl acetate; (b) recovering the extracted cocoa solids;(c) extracting the recovered extracted cocoa solids with a solventselected from the group consisting of acetone and ethanol and mixturesthere of with up to 50% water, which solvent is optionally acidified toa pH of about 2 to about 4 with an edible acid; (d) separating the cocoasolids from the cocoa extract; and (e) optionally drying the cocoaextract.
 27. The method of claim 26, wherein the cocoa beans areunfermented or underfermented.
 28. The method of claim 27, wherein theextraction is carried out at about 20° C. to about 50° C.
 29. The methodof claim 26, wherein the higher oligomers include at least tetramers andabove.
 30. A cocoa extract prepared by the process of claim
 27. 31. Acocoa extract consisting essentially of cocoa procyanidin tetramersthrough at least decamers.
 32. A continuous method for extracting cocoapolyphenols including cocoa procyanidins from partially defatted orfully defatted cocoa solids using an organic solvent suitable forsolubilizing cocoa procyanidins, or mixtures thereof with water, whichcomprises the steps of (a) contacting a series of cells containing thecocoa solids with the solvent flowing in a counter-current direction,(b) recovering the cocoa extract, and (c) drying the cocoa extract. 33.The process of claim 32, wherein the cocoa beans are unfermented orunderfermented cocoa beans.
 34. The process of claim 32, wherein theprocess is carried out under pressure.
 35. The process of claim 32,wherein the solvent is a lower alkyl alcohol, a lower alkyl ketone, or alower alkyl acetate.
 36. The process of claim 33, wherein the loweralkyl alcohol is methanol, ethanol, or isopropanol, the lower alkylketone is acetone, and the lower alkyl acetate is methyl acetate orethyl acetate.
 37. The process of claim 32, wherein the cocoa extract isrecycled until substantially no more cocoa procyanidins are extractedfrom the cocoa solids.
 38. The process of claim 32, wherein the solventis the organic solvent and the extract is ried by flashing off thesolvent.
 39. The process of claim 32, wherein the solvent is theaqueous-organic mixture and the extract is dried by flashing off thesolvent before drying the aqueous extract.
 40. The process of claim 32,wherein the drying of the aqueous extract is carried out by freezedrying, spray drying, flash drying, or drum drying.
 41. A method forrecovering cocoa procyanidins and theobromine from partially defatted orfully defatted cocoa solids, prepared from unroasted cocoa beans,comprises the steps of (a) extracting the cocoa solids with ethanol atabout 50° to about 70° C., (b) chilling the ethanol extract at atemperature and for a time sufficient to precipitate out thetheobromine, (c) separating the precipitated out theobromine, and (d)removing the ethanol from extract to recover the dried cocoaprocyanidins.
 42. A method of minimizing the amount of theobromine inthe cocoa procyanidins extracted from partially defatted or fullydefatted cocoa solids, prepared from unroasted cocoa beans, comprisesthe steps of (a) extracting the cocoa solids with isopropanol to producean alcoholic extract containing cocoa procyanidins and theobromine, (b)chilling the alcoholic extract at a temperature and for a timesufficient to precipitate out the theobromine, and (c) separating thetheobromine from the alcoholic extract.
 43. The method of claim 41 or42, wherein the chilling is carried out for about 1 hour to about 12hours at 0° to about 5° C.
 44. The method of claim 41 or 42, wherein theseparating step is carried out by filtering the extract, centrifuging,the extract, or by an adsorbing the theobromine on a substrate.